Lymphangiogenesis is the formation of new lymphatic vessels from preexisting lymphatic vessels and is associated with diverse pathological conditions including metastatic dissemination, graft rejection (e.g. cornea, kidney and heart), type 2 diabetes, obesity, hypertension, and lymphedema (Alitalo, K., et al. 2005. Nature 438:946-953; Karaman, S., et al. 2014. J Clin Invest 124:922-928; Kim, H., et al. 2014. J Clin Invest 124:936-942; Maby-El Hajjami, H., et al. 2008. Histochem Cell Biol 130:1063-107; Machnik, A., et al. 2009. Nat Med 15:545-552; Mortimer, P. S., et al. 2014. J Clin Invest 124:915-921; Skobe, M., et al. 2009. Nat Med 15:993-994).
Lymphatic vessel invasion in and around a primary tumor compared to invasion of blood vessels is a prognostic marker of the aggressiveness of various types of cancers. Growth of lymphatic vessels is also involved in graft rejection (Dietrich, T., et al. 2010. J Immunol 184:535-539; Hall, F. T., et al. 2003. Arch Otolaryngol Head Neck Surg 129:716-719; Maula, S. M., et al. 2003. Cancer Res 63:1920-1926; Miyata, Y., et al. 2006. J Urol 176:348-353; Saad, R. S., et al. 2006. Mod Pathol 19:1317-1323; Schoppmann, S. F., et al. 2004. Ann Surg 240:306-312; Zeng, Y., et al. 2005. Prostate 65:222-230).
A regulatory protein that induces lymphangiogenesis is a tyrosine kinase receptor identified as vascular endothelial growth factor receptor-3 (VEGFR-3), which is highly expressed in lymphatic endothelial cells (LECs) and is further upregulated in response to inflammation (Flister, M. J., et al. 2010. Blood 115:418-429). That VEGFR-3 plays a central role in lymphangiogenesis has been demonstrated by showing a reduction in lymphatic vessel density following VEGFR-3 blockade during a variety of pathological conditions including malignancy and chronic inflammation (Matsumoto, M., et al. 2013. Clin Exp Metastasis 30:819-832; Pytowski, B., et al. 2005. J Natl Cancer Inst 97:14-21; Singh, N., et al. 2013. Blood 121:4242-4249).
Podoplanin (PDPN), first detected on the surface of podocytes, is a transmembrane receptor protein (Breiteneder-Geleff, S., et al. 1997. Am J Pathol 151:1141-1152). PDPN is expressed by LECs but not by blood endothelial cells and promotes blood-lymph separation during development. Mice lacking PDPN have leaky lymphatic vessels and congenital lymphedema (Fu, J., et al. 2008. J Clin Invest 118:3725-3737; Schacht, V., et al. 2003. Embo J 22:3546-3556). PDPN expression in LECs is required for lymphatic capillary tubule formation in matrigel a VEGF-A-induced cell migration in scratch wound assays (Navarro, A., et al. 2008. Am J Physiol Lung Cell Mol Physiol 295:L543-551; Navarro, A., et al. 2011. Am J Physiol Lung Cell Mol Physiol 300:L32-42). The extracellular domain of PDPN plays a critical role in lymphangiogenesis. The Fc fusion protein of PDPN extracellular domain (PDPN-Fc) inhibits LEC migration and tube formation in cell culture and suppresses lymphangiogenesis but not hemangiogenesis in inflamed mouse corneas in vivo (Cueni, L. N., et al. 2010. Blood 116:4376-4384). These results indicate a requirement of PDPN in lymphangiogenic process. The extracellular domain of PDPN is heavily glycosylated, and O-glycosylation and sialylation are involved in PDPN-mediated blood-lymph separation and platelet aggregation (Fu, J., et al. 2008. J Clin Invest 118:3725-3737; Kaneko, M. K., et al. 2007. FEBS Lett 581:331-336).
Members of the galectin family of mammalian lectins characterized by a carbohydrate recognition domain (CRD) with affinity for β-galactoside containing glycans, play a role in hemangiogenesis. A galectin family member, Gal-3, modulates VEGF-induced angiogenic response by binding through its CRD to the N-glycans of αvβ3 integrin and VEGFR-2 and subsequently activating angiogenic signaling pathways (Markowska, A. I., et al. 2011. J Biol Chem 286:29913-29921; Markowska, A. I., et al. 2010. J Exp Med 207:1981-1993). VEGFR-3 and PDPN are glycosylated similarly to most cell receptors. However, little is known about the function of the galectins in the context of the carbohydrate-mediated recognition system. Gal-8 is a tandem-repeat type member of galectin family and contains two different CRDs. The N-terminal CRD (Gal-8N) prefers α2,3-sialyl glycans and mainly contributes to unique carbohydrate-binding specificity of this protein (Carlsson, S., et al. 2007. Glycobiology 17:663-676; Ideo, H., et al. 2011. J Biol Chem 286:11346-11355; Ideo, H., et al. 2003. Glycobiology 13:713-723). Gal-8 is robustly expressed by LECs, binds to PDPN, and promotes LEC haptotactic migration when immobilized on to a surface (Cueni, L. N., et al. 2009. Exp Cell Res 315:1715-1723 and Detmar, 2009).
Anti-lymphangiogenic agents are useful for treatment of debilitating diseases of the eye. The growth of lymphatic vessels is the major reason of corneal graft rejection (Dietrich, T., et al. 2010. J Immunol 184:535-539). Penetrating keratoplasty is the most common form of solid tissue transplantation. Approximately 40,000 corneal transplantations are performed each year in the United States. Success rate of penetrating keratoplasty is as high as 90% for uncomplicated first grafts performed in avascular low-risk beds. However, the rejection rate of the corneal grafts placed in high-risk vascularized host beds is extremely high (70% to 90%). Thus the development of safe and targeted new regimens to inhibit lymphangiogenesis is needed to promote graft survival.
Anti-lymphangiogenesis drugs are useful also for treatment of dry eye disease. Significant upregulation of prolymphangiogenic factors (e.g. VEGF-C, VEGF-D, and VEGFR-3) and selective growth of lymphatic vessels without concurrent growth of blood vessels has been demonstrated in corneas with dry eye disease (Goyal, S., et al. 2010. Arch Ophthalmol 128:819-824). Dry eye disease is an immune-mediated disorder affecting about 5 million Americans. It severely impacts the vision-related quality of life and the symptoms can be debilitating. The current therapeutic options for dry eye disease are limited, mostly palliative, and expensive. Therefore, development of lymphangiogenesis inhibitors is of therapeutic value for treatment of dry eye disease.
Moreover, there is need for development of pro-lymphangiogenic therapy for disorders such as lymphedema, a condition of localized fluid retention caused by a compromised lymphatic system. Lymphedema affects approximately 140 million people worldwide (Brorson, H., et al. 2008. Lymphology 41:52-63). The disease most frequently occurs after surgical removal of lymph nodes or radiation therapy, during the treatment of cancer. It is a progressive and lifelong complication notably of breast cancer for which no curative treatment exists (Szuba, A., et al. 1998. Vasc Med 3:145-156; Witte, M. H., et al. 2011. J Surg Oncol 103:489-500). Despite the well-established significance of lymphatics in the pathogenesis of numerous diseases, little is known about effective anti-lymphangiogenic agents compared to the abundance of anti-hemangiogenic agents that have entered clinical trials. Therefore, development of lymphangiogenesis inhibitors is of therapeutic value for treatment for lymphedema.
There is a need for methods and compositions that inhibit the activities of pro-lymphangiogenic factors and for methods that prevent or treat graft rejection, dry-eye disease tumor metastasis, lymphedema and other inflammatory conditions.